This goal of this proposal is to investigate the best experimental and computational approaches for assigning hydrogen atom positions in protein crystal structures. Hydrogen is arguably the most important atom in proteins, yet it is virtually invisible in nearly all protein crystal structures because they are typically determined at too low a resolution (even at very high resolution not all of them are visible, for unknown reasons). We will create a "gold standard" set of protein crystal structures at ultra-high resolution as a function of different pH values, and will compare those results with a similar series of neutron diffraction structures on the same proteins, where the hydrogen atoms can be observed directly. These structures will allow us to devise improved computational and experimental procedures to locate hydrogens in structures at less than optimal resolutions. Moreover, these studies will be performed at both cryogenic and room temperatures, thus enabling for the first time, the determination of the effects of cryoprotection and flashcooling on the ionization states of critical protein side chains.